Na+ channels are highly clustered at the neuromuscular junction (NMJ) deep in the junctional folds below acetylcholine receptors (AchRs). Whereas AchRs respond to the release of acetylcholine from the motor neuron and are responsible for the initial membrane depolarization, the clustered Na+ channels are responsible for the muscle action potential. AchR clustering depends on a combination of agrin, neuregulin, and activity dependent mechanisms that have been described in great detail. However, little is known about the mechanisms responsible for NMJ Na+ channel clustering. Diseases including myotonia, periodic paralysis, and myasthenic syndrome all disrupt the NMJ. Na+ channel clustering occurs at two other locations in the nervous system including nodes of Ranvier and axon initial segments (AIS). Here, both cytoskeletal and extracellular interactions participate in channel clustering and the mechanisms have been described in detail. Remarkably, many of the same proteins involved in Na+ channel clustering at nodes and AIS are also found at the NMJ. By analogy to nodes and AIS, we propose that NMJ Na+ channel clustering depends on similar cytoskeletal and extracellular interactions. Aim 1 will consist of two parts designed to determine the cytoskeletal interactions important for NMJ Na+ channel clustering. First, we will conditionally knockout (specifically in skeletal muscle) the three Na+ channel-binding ankyrins (Ank1-3) singly and in combination. Second, we will conditionally knockout the 4 different spectrins known to be expressed in muscle and that are thought to link ankyrins (and Na+ channels) to the actin cytoskeleton. In both ankyrin and spectrin deficient mice we will evaluate muscle function and Na+ channel clustering. In Aim 2 we will identify the extracellular interactions that participate in NMJ Na+ channel clustering. First, we will generate muscle-specific knockouts of the cell adhesion molecule Nfasc given its location at the NMJ and its important role mediating extracellular interactions at the AIS and nodes. Second, since much less is known about the cell adhesion molecules and extracellular matrix molecules that may underlie NMJ extracellular interactions, we will use proximity biotinylation methods and proteomics to identify these proteins. We will then validate potential candidates for their localization to the NMJ, and using gain and loss of function strategies determine their functions. The aims proposed here will dramatically improve our understanding of the molecular mechanisms controlling Na+ channel clustering at the neuromuscular junction and may lead to important insights into the pathophysiology of neuromuscular diseases and neuropathies where NMJs degenerate or function is compromised.